Glycolipids are glycosyl derivatives of lipids such as acylglycerols, ceramids and prenols. They are components of cell membranes found in species ranging from bacteria to man. Because of the diversified structures, glycolipids perform a variety of functions in living organisms (Curatolo, W. Biochim. Biophys. Acta, 906, 1987, 111–136). In recent years, a wide number of new glycolipids have been synthesized or isolated from natural sources. The role of glycolipids in various organisms, organs, tissues and membranes is currently attracting a lot of attention (Curatolo, W. Biochim. Biophys Acta, 906, 1987, 137–160).
The physiological functions of glycolipids are yet to be understood completely. It appears that glycolipids serve four general functions in cell membranes; stablilization, shape determination, recognition and ion-binding (Curatolo, W: Bichem. Biophys. Acta, 906, 1987, 137–160). Certain glycolipids carry out similar functions whether they are observed in bacteria, plants or animals. Some of these are:                1. Impart structural integrity and decreased permeability to myelin membranes in mammalian brains (Oldfield, E. and Chapman, D, FEBS Lett., 21, 1972, 393–306, Ladbrooke, B. D., et al, Biochim. Biophys. Acta, 164, 1968, 101–109).        2. Provide stabilization to the brush border membranes intestinal epithelium (Hauser, H., et al, Biochim, Biophys, Acta, 610, 1980, 567–577; Br4cimer, M. E., et al, J. Biol, Cihem, 257, 1982, 557–568; Brasitus, T. A., and Schachter, D. Biochemistry, 19, 1980, 2763–2769); also membranes of the tubules of kidney (Karisson, K. A., et al, Biochim. Biophys. Acta, 316, 1973, 317–335; Tomono, Y., et al, Biochim. Biophys. Acta. 796, 1984, 1999–204) and human colon (Corfield, A. P. et al, Biochem, Soc., Trans, 19(2), 1991, 220).        3. Act as a permeability barrier of stratum corneum, the outer most layer of the skin (Gray, et al, Br. J. Dermatol, 106, 1982, 59–63); and        4. Function are receptors for virus and bacteria (Haywood, A. M. J. Mol. Biol, 87, 1974, 625–628), carbohydrate binding proteins, plant and animal lectins (Surolin, A., et. al, Nature, 257, 1975, 802–804; Rando, R. R. and Bangerter, F. W., J. Supramol. Struct. 11, 1979, 295–309; Wassef, N. M. et al, Biochem. Biophys. Res. Commun. 130, 1985, 76–83), bacterial toxins including cholera toxins (Van Heyningen, S. Science, 183, 1974, 656 657), tetanus toxins (Van Coli entereotonins (Moss, J., et. al, J. Biol, Chem, 256, 1981, 12861–12865) etc. and hormones (Mullin, B. R. et al, Proc. Natl. Acad. Sci. USA, 73, 1976, 842–846; Becker, S. K. et al., Proc. Natl. Acad. Sci. USA, 78, 1981 4848–4852, Fishman, P. H., et al. J. Biol. Chem. 250, 1984, 7983–7989).        
Glycolipids play an important role in human health. It has been reported that qualitative and quantitative changes in the glycolipids of cells occur during differentiation and oncogensis (Hakomori, S. Biochim. Biophysm Acta, 417, 1975, 55 89; Feizi, T., Nature, 314, 1985, 53–57). Extensive analysis of the glycolipids of tumorous tissues showed both quantitive and quatitative differences with normal tissues (Hamomori, S. and Murakami, W. T., Proc. Natl. Acad. Sci., USA, 59, 1968, 254–261). Antibodies were used to characterise a variety of tumor antigens which were carried on gangliosides (Tai, T., et al., Biochim. Biophys, Acta, 835, 1985, 577–583) and neutral glycosphingolipids (Willison, K. R., et al. J. Biol. Chem, 1983, 4091–4097). These extensive studies of glycolipid antigens on cell surfaces have provide wealth of information related to glycolipid synthesis and the nature of the cell surface in differentiation and oncogenesis.
Glycolipids have been implicated in a variety of immunological phenomena. The possible involvement of gangliosides in the action of interferon was reported long back (Bensancon, F. and Ankel, I I Nature, 252, 1974, 478–480). Interleukin-2 was shown to have affinity towards gangliosides (Parker, J., et al, FEBS Lett., 170, 1984, 391–395). However, the area still remains wide open as another group of scientist showed that although gangliosides binds interleukin-2 in vitro, it is unlikely that these glycolipids act as physiologically relevant interleukin-2 receptors (Robb, R. J. Immumol, 136, 1986, 971–976). Involvement of glycolipids in lymphocyte stimulation has been suggested by a series of studies (Spiegel, S., et al., Proc. Natl. Acad. Sci., 76, 1979, 5277–5281; Spiegel, S and Wilchesk, M. J. Immunol, 127, 1981, 572–575; Spiegel, S. and Wilcheck, M., Mol. Cell. Biochem. 55, 1983, 183–190). It was also indicated that the ganglisides may be involved in immuno regulations (Miller, H. C. and Essenman, W. J. J. Immunol. 115, 1975, 839–843); Chaney, W. G. et. al., Cell Immunol. 86, 1984, 165–170). Inhibitation of concanavalin 4-induced mitogentic response in mouse thymocytes by the gangliosides was demonstrated (Langle, E. E. et al., Cancer Research, 39, 1979, 817–822). In another communication, possible immunosuppressive effect of gangliosides shed from tumor cells was indicated (Ladish, S, et. al., Cancer Research, 43, 1983, 3808–3813).
The involvement of gangliosides in the growth of neutrons is well established. Extensive proliferation of neutires of felines with gangliosidosis was observed (Purpura, D. P. and Baker, H. J Brain Res., 143, 1978, 13–26). These effects could be elicited by a variety of purified gangloslides (Byrene, M. C., et al, J. Neurochem., 41, 1983, 1214–1222). The promising in vitro results was followed by attempts to cure neuropathies with ganglioside injections (Gorio, A. et al, in Ganglioside Structure, Function and Biomedical Potential, Ed. by Ledeen, R, Yu, R, Raport, M and Suzuki, K. Plenum Press, New York, 1984, 549–561). Initial clinical trials on humans indicated that gangliosides injections may improve symptoms of diabetic neuropathy (Narden, A., et al., in Ganglioside Structure, Functions and Biomedical Potential, ed. By Ledeen, R, Yu, R., Rapoort, M and Suzuki, K, Plenum Press, New York, 1984, 593–600).
The mechanism of the actions of various glycolipids in regulating physiological function are yet to be understood and in recent years, extensive studies are being carried out throughout the world on the biological activities of glycolipids isolated from various natural resources as well as of synthetic glycolipids. The use of glycolipids in pharmaceurical and cosmetic preparations is increasing at a brisk pace.
In a recent communication, possible use glycodlipids from sivers sagebrush (wormwood) as organoleptic and biologically active additive in food industries were evaluated (Gubanenko, G. A., et al, Pischch. Prom. St. 6, 1998, 26–27). Glyocolipids are implicated for many physiological functions and based on these findings, newer drugs were being formulated. Cell uptake and transfection efficiency of DNA/glycolipid complexes were implicated as potential HIV-1 fusion cofactor by a group of scientists (Djilalj, H., et al, Biochim, Biophys. Res. Commun. 246(1), 1998, 117–122). U.S. Pat. No. 5,871,714 discloses the use of glycolipid-based compositions for controlling of colonization of bacterial plaque in the oral cavity (Bundy, J. A. 1999). PCT Int. Appl. WO 99,00136 discloses another formulation comprising of glycolipids for use in treatment or prohylaxis or acidic gut syndrome resulting from accumulation of acids and production of endotoxins in the gastro-intestinal tract (Rowe, J. B., 1999). The role of glycolipids on age related changes of the brain and also in Alzheimer's disease was reviewed recently (Endo, T. Tampakskitu Kakusan Koso, 43 (16), 1998, 2582–2588). In another communication, the role of glycolipids in type 1 diabetes and lupus, in intracellular bacterial infections and in tumor rejections were reviewed (Park, S. II. et al, Semin. Immunol., 10(5), 1998, 391–398). Structural features and biological activities of some natural and synthetic glycolipid antigens which induce a CD-1 restricted T-cell response are discussed in PCT Int, Appl. WO 99,12562 (Porcelli, S. A. and Moody, D. B., 1999). These glycolopids can be used for treatment of individuals infected with Mycobacterium leparae. Glycolipids were found to have an important role in lens fiber developed in human eye (Ogiso, M, Acta, Biochim. Pol., 45 (20, 1998, 501–507).
Sophorolipids are very important glycolipids which can be isolated from various sources (Marchal, R., et al., U.S. Pat. No. 5,900,366, 1999: Daniel; H. S. et al, Biotechnol., 51 (1), 1999, 40–45). In one investigation, sophorolipid was produced from deproteinized whey and rapessed oil (Daniel, H. S., et al, Biotechnol, Lett, 29(12), 1998, 1153–1156). Bioactivity of the extracellular glycolipids like sophorolipids and sophorolipid-derivatives were investigated (Sholtz., C., et al., Polym, Prepr. 39 (2), 1998, 168–169). These glycolipids were found to exhibit cell growth inhibition properties for Jurkat (Leukemia) and Tu-138 (head and neck cancer) cells. Glycosylphosphotidyl inositols represent predominant class of glycolipids synthesized by the asexual intra-erythrocytic stages of Plasmodium falciparum. These glycolipids have an effect on material toxins and in release of cytokines like tumor necrosis factor- and interleukin-10 (Schmidt, A., et al, Exp. Parasitol., 88(2), 1998, 95–102).
Japanese scientists have isolated a new glycolipid from Gigartina tenella (Ohra K, et al., Jpn. Kokai Tokkyo Koho, JP 11, 106,395, 1999). This glycolipid from red seawood was found to be highly active as DNA-synthetase B-inhibitor, HIV inhibitor and as immunosuppressant. This was also found to act as anti-cancer agents along with some other immunosuppressive effects (Yoshida, M., et al., Jpn Kokai Tokkyo Koho JP, 10, 152,498, 1998). Clusters of glycolipids and glycophosphotidyl inositol-anchor proteins in lymphoid cells were investigated for their cellular responses to raft patch formation in the Jurkal (Leukemia) T-cell lines and, in particular, changes in the actin cytoskeleton (Harder, T. and Simons, K., Eur., J. Immunol, 29 (2), 1999, 556–562). Growth factor-induced release of a glycosyl-phosphatidyl inositol (GPI)-linked protein from HEP-2 human carcinoma cell lines was studied by a group of English scientists (Roberts, J. M., et, al., FEBBS Lett., 267 (2), 267 (2), 1996, 213–216). The suitability of glycolipids in gene therapy was also tested by a group of scientists (Havermann, K., et. al, EP 893,493, 1999).
Glycolipids are excellent surface-active agents. In fact, a majority of naturally occurring surfactants (bio-surfactants) are glycolipids. However, bio-sufactants are fermentation products and their commercial productions, at present, are not economically feasible. In context, their isolation from cheap natural products such as rice bran oil assumes importance. A few of the glycolipids isolated from other natural resources have been tested for then surface active properties. Because of the complete biodegradability, these are being evaluated for special uses. Glycolipids were used as synergists in a detergent formulation made for manual dish-washing (Udo, H., et, al., Ger. Offen. DE 19,648,439, 1998). Glyolipid based biosurfactant complexes, produced by micro-organisms of Rhodococcus species was used for oil desorption from minerals and organic materials (Ivashina, I. B., et, al., J. Microbiol. Biotechnol, 14(5), 1998, 711–717). Another glycolipid type of biosurfactant was obtained from a strain of Pseudomonus Aerugmosa isolated from soil (Cho, J. H., et al., J. Mirobiol. Biotechnol, 8(6), 1998, 645–649). Critical microllar concentration of this surfactant was found to be very low—as low as 50 ppm and the minimum surface tension was obtained was 30.1 mN/m. It showed very good emulsifying property, better than a well known emulsifier like emulsan. Foaming power and emulsifying properties of some other glycolipid-based biosurfactants isolated from pumpkin were evaluated (Nakac, T., et al., Food Sci, Technol. Int. 4(3), 1998, 235–240). These surfactants also showed good foaming power and emulsifying properties comparable to any commercial surfactant. A group of German scientists have described a method for the microbial production of surface active glycolipids from vegetable oil and carbohydrates (Lang, S., et al, Food Sci, Technol. Int., 4(3), 1998, 235–240). These surfactants also showed good foaming power and emulsifying properties comparable to any commercial surfactant. A group of German scientists have described a method for the microbial production of surface active glycolipids from vegetable oils and carbohydrates (Lang, S., et al., Schrifter. Nachwachsende Rohst, 10, 1998, 154–163). The glycolipid formed by this method reduced the surface tension of water from 72 to 32 mN/m. Rhamnolipids and sophrolipids are two glycolipids that show considerably high degree of surface activities. Recently a facile procedure for remediation of oily with rhamnolipid biosurfactant was reported(Nakata, K. and Ishiggami, Y., J. Environ, Sci, Health—Part A: Toxic/Hazard. Subst. Environ. Eng. A 34(5), 1999, 1129–1142). A simple remediation process for mousse oil wastes was carried out using rhamnolipid as bioemulsifier. The feasibility of using biosurfactants to remove heavy metals from an oil contaminated soil was evaluated by hatch washes with surfacing, a rhamnolipid and a sophorolipid and the results obtained were quite encouraging (Mulligan, C. N., et. al., Environ. Prog., 18(1), 1999, 50–54).
With such wide ranging nature, functions and biological activities, it is surprising that these compounds are not exploited commercially and great extent. Possible reasons for this could be the lack of availability of natural sources rich in these glycolipids or commercially viable processes for their isolation. Rice bran oil appears to offer significant possibilities on these count glycolipids content is quite high. These glycolipids contain a number of compounds of wide structure complexities including a novel phosphorus containing glycolipid identified by the present authors (unpublished) and these can be isolated by simple, commercially processes as detailed in the present invention.
India is the second largest producer of rice (after China). About 2.8 million tons of rice bran is processed in India every year yielding about 500,000 tons of rice bran oil. Crude rice bran oil of Indian origin contains 2–3.5% of waxes, 1–2% of gums (phosphatides) and 2.5–3.5% of glycolipids (Kyong-Soohm, et al, Han'guk Sikp'um Younygang Kwahack Hocchi, 25(5), 1996, 735–740). To get a better quality of edible oil as well as for the smooth operation of the later processing steps, the removal of both waxes and gums are necessary. Dewaxing is generally done at the final stages of refining by the process of winterization whereas degumming is done before alkali neutralization.
Crude rice bran oil shows an unusual behaviour as compared to the other vegetable oils. It can hold its own volume of hot water without the water separating out from the oil This property was taken advantages of in developing a process for simultaneous degumming and dewaxing of the oil (Kaimal, T. N. B., et al., Indian Patent No. 183639, 2000). This particular property was also not observed in high phospholipid containing oil like soybean oil and wax containing oil like sunflower oil and probably arises from the high content of glycolipids in the rice bran oil. Moreover, in contrast to other vegetable oils, rice bran oil contains a number of special types of glycolipids. A series of steryl glycosides (Fujino. Y. et al., Biochim. Biophys. Acta, 574, 1979, 94), ceramides and ceramide monohexocides were reported in the bran (Fujino, Y. et al, J. Food Sci., 39, 1974, 471; Fujino, Y. et al, Chem, Phys, of Lipids, 17, 1976, 275) and different glyceroglycolipids (Sasyty, P. S. et al., Biochemistry, 3, 1964, 1271; Miyano, M., et al., J. Am. Oil Chem. Soc., 57, 1962, 84) have also been reported in rice bran. The water absorbing capacity of rice bran oil may be attributed to the presence of these special types of glycolipids. The sludge generated in degumming/dewaxing of rice bran oil was found to contain the whole amount of water used in the degumming/dewaxing operation in a smooth emulsified form and this also may be attributed to the presence of mixture of glycolipids like ceramides, sphingolipids, steryl glycosides etc. The emulsion obtained was very strong and was strong and was stable for a long time. The analysis of that sludge, indeed shoed the presence of higher amount of glycolipids. It is well documented that not only in the bran but also in the rice bran oil the glycolipid content is very high compared to the other oils. It was also reported that the glycolipids fraction of rice bran oil contains a mixture of different types of glycosides, glyceroglycolipids, sphingolipids and ceramides group of compound (Nasirullah and Nagaraia, K. V., J. Oil Tech, Asso, India, 19, 1987, 2; Kyong-Soohn, et al, Han'guk Sikp'um Yonygang Kwahak Hoechi, 25(5), 1996, 735; Fujino, Y. and Ohnishi, M., Chem. And Phys. Of Lipids, 17, 1976, 275).
These glycolipids, if isolated, can be used in various foods, pharmaceuticals and nutraceutical formulations. These glycolipids may find their uses in cosmetics industries as well. It is widely accepted that the rice bran oil has many nutritional and health benefits. This is attributed mainly to γ-oryzanol, a constituent of rice bran oil. However, the nutritional aspects of the novel glycolipids present in rice bran oil are yet to be investigated and these glycolipids may also contribute to the extraordinary health benefits shown by rice bran oil.
A few attempts have also been to utilize the various glycolipids present in rice bran and rice bran oil. In recent patent (Jpn. Kokai Tokkyo Koho, JP 11,113,530, 1999), it was claimed that the ceramides extracted from rice bran can be utilized to prepare a formulation which shows skin-moisturizing, rough skin-preventing and anti-wrinkle effects. In another patent (Ger. Offen. DE 4,130,915, 1992), sphingolipids were used as an important ingredient in a scalp and skin-moisturizer formulation. It was also observed that the sphingolipids could be combined with conventional ingredients to provide a shampoo that inhibits dandruff formation by more than 50%. The added advantages of these molecules are they show very high activity even at a very low concentration. Rice bran extracts reportedly were used in the preparation of hair growth stimulants (South African Patent ZA 92,00186, 1992). Significant amount of ceramides and sphingolipids are present in rice bran (Fujino, Y. and Ohnishi, M Chem. Phys. of Lipids, 17, 1976, 275). These sphingolipids, ceramides and their derivatives may also be used for the same purpose. A bath preparation that prevents itching and eczema of the skin was patented (Jpn. Kokai Tokkyo Koho, JP 09, 118,614, 1997) which has ceramides as an important ingredient. Apart from the examples cited above, there exist a number of cosmetic formulations, which are based on rice bran oil extracts or rice bran oil itself (Jpn, Kokai Tokkyo Koho, JP 09, 12, 443, 1997; Jpn. Kokai Tokkyo Koho, JP 06,345,635, 1994; Jpn. Kokai Tokkyo Koho, JP 02,264,706, 1990; Jpn. Kokai Tokkyo Koho, JP 11,05,730, 1999). In all these processes, the glycolipids used in the formulations were isolated from rice bran or the other sources by using the conventional methods.
Glycolipids are conveniently isolated in the laboratory by (I) extraction of the total lipids with CHCl3/CII3OII (2:1 v/v) and water saturated butanol and (ii) silicic acid column chromatography employing sequential elution with CHCl3, acetone and CH3OH (Fujino, Y. and Ohnishi, M. Chem. Phys. of Lipids, 17, 1976, 275) to yield three fractions containing neutral lipids, glycolipids and phospholipids respectively (Rouser, G. et al, Lipids, 2, 1967, 37). This method has also been followed to isolate glycolipids from rice bran oil (Fugino, Y. et al, Biochim, Biophys. Acta, 574, 1979, 94). This laboratory method, however, is not easily adapted by the industry because the method requires large amount of solvents and it is time-consuming as well. In view of these difficulties, no serious efforts were made to isolate the glycolipids present in the rice bran oil for commercial explorations. Japanese scientists, in an alternative way of isolation, used latrobeads column to recover glycolipids from soy protein isolates (Homma, S. and Murata, M Darzu Tanpakushitur Kenkyakai Kaishi, 14, 1993, 104). In a more recent communication, a method to isolate a specific glycolipid from boar spermatozoa using ion-exchange chromatography in combination with partition chromatography was described (Iga, D. P., et. al., Bio, 45, 1996, 9). The major drawbacks of these processes are that these are expensive and time consuming.